Soybean transformation method

ABSTRACT

A method is disclosed for the  Agrobacterium -mediated germline genetic transformation of soybean. The method is based on  Agrobacterium -mediated gene delivery to individual cells in a freshly germinated soybean meristem, which cells can be induced directly to form shoots that give rise to transgenic plants. This method does not involve callus-phase tissue culture and is rapid and efficient.

This application claims priority to US provisional application60/115,833 filed Jan. 14, 1999, and U.S. patent application Ser. No.09/483,472 filed Jan. 14, 2000 incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to plant cell transformation andregeneration into a differentiated transformed plant. More particularly,the invention relates to a method for transforming soybean (Glycine max)using Agrobacterium-mediated transformation of a plant tissue explantand subsequent regeneration of the transformed cells into a whole plant.

BACKGROUND OF THE INVENTION

Cultivated soybean (Glycine max) has a substantial commercial valuethroughout the world. Over 50 million hectares worldwide are used toproduce an annual crop of soybeans in excess of 100 metric tons with anestimated value exceeding 20 billion dollars. The development ofscientific methods useful in improving the quantity and quality of thiscrop is, therefore, of significant commercial interest.

Modern biotechnological research and development has provided usefultechniques for the improvement of agricultural products by plant geneticengineering. Plant genetic engineering involves the transfer of adesired gene or genes into the inheritable germline of crop plants suchthat those genes can be bred into or among the elite varieties used inmodern agriculture. Gene transfer techniques allow the development ofnew classes of elite crop varieties with improved disease resistance,herbicide tolerance, and increased nutritional value. Various methodshave been developed for transferring genes into plant tissues includinghigh velocity microprojection, microinjection, electroporation, directDNA uptake, and Agrobacterium-mediated gene transformation.

Agrobacterium-mediated gene transformation is the most widely used genetransfer technique in plants. This technique takes advantage of thepathogenicity of the soil dwelling bacteria, Agrobacterium tumefaciensor Agrobacterium rhizogenes. Agrobacterium tumefaciens natively has theability to transfer a portion of its DNA, called T-DNA, into the genomeof the cells of a plant to induce those cells to produce metabolitesuseful for the bacterium's nutrition. Agrobacterium-mediatedtransformation takes advantage of this concept by replacing the T-DNA ofan Agrobacterium with a foreign set of genes, thus, making the bacteriuma vector capable of transferring the foreign genes into the genome ofthe plant cell. Typically, the foreign gene construct that istransferred into the plant cell involves a specific gene of interest,which is desired to be introduced into the germiline of the plant,coupled with a selectable marker that confers upon the plant cell aresistance to a chemical selection agent. Typically, theAgrobacterium-mediated gene transfer is into an undifferentiated cellcultivated in tissue culture, known as a callus cell, or the transfer ismade into a differentiated plant cell from a leaf or stem, which is theninduced to become an undifferentiated callus culture.

Although significant advances have been made in the field ofAgrobacterium-mediated transformation methods, a need continues to existfor improved methods to facilitate the ease, speed and efficiency ofsuch methods for transformation of soybean plants.

SUMMARY OF THE INVENTION

The present invention provides a novel and efficient method ofperforming germline transformation of soybean usingAgrobacterium-mediated transformation directly on meristematic cells ofsoybean embryos. Direct shoot induction from transformed meristematiccells results in germline transgenic plants. The overall process israpid and efficient.

One significant aspect of this invention is that the reduction of thepretreatment period of soybean seeds has improved the shoot productionin surviving explants as well as reduced the time taken to produceplants that are transferable to a greenhouse. Also, the reduction oftime and materials provides a system that is economically beneficial tothose who implement it.

It is an object of the invention to provide a rapid and efficient methodto perform soybean genetic transformation using Agrobacterium-mediatedgene transfer.

It is another object of the present invention to provide a soybeantransformation method not requiring a step of callus culture so that themethod can be used on any soybean variety.

Another aspect of the present invention is to provide novel methods ofwounding to increase transformation efficiency. One method of woundinginvolved exposing soybean embryos to ultrasonic sound waves (i.e.,sonication). Another method involves wounding via a plasma blast of anelectric gene gun.

Other objects, advantages, and features of the present invention willbecome apparent from the following specification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method for the direct germline genetictransformation of varieties of soybean, Glycine max. This method isbased on Agrobacterium-mediated gene delivery into individual soybeancells in the meristem of a soybean embryo. The transformed cells arethen induced to form shoots that are, at a high frequency, germlinesoybean transformants that can be cultivated into whole sexually matureand fertile transgenic soybean plants. The method does not involve aphase of callus culture, and hence the time period of the entire processfrom seed to transgenic seed is remarkably concise.

The method described here is based on Agrobacterium-mediated genedelivery into growing cells in an embryonic meristem.Agrobacterium-mediated techniques typically only result in gene deliveryinto one, or only a few, cells in the targeted tissue. Typically, aselective agent is applied post-transformation to kill all of the cellsin the targeted tissues that are not transformed or to identifytransformed cells through a selective advantage. Then a callus or otherproliferative growth of transformed cells can be grown from which plantscan be ultimately regenerated.

The method described here does not utilize a callus or proliferativephase. Instead, the Agrobacterium-mediated gene delivery is made intocells in the living meristem of a soybean embryo excised from a soybeanseed. Then the meristematic region is cultured in the presence of aselection agent and a hormone to induce direct shoot formation.Preferably, the meristem is cultivated in the presence of the herbicideglyphosate, which acts both as a selection agent as well as ashoot-inducing hormone. The result of this step is the termination or atleast growth retardation of most of the cells into which the foreigngenetic construction has not been delivered and the simultaneousinduction of the formation of soybean shoots, which arise from a smallcluster of cells including a transformed meristematic cell. The meristemcan also be cultivated in the presence of a selection agent, including,but not limited to, kanamycin.

This method is cultivar independent. The soybean tissue manipulations inthis process are analogous to those in prior particle-mediatedtransformation methods, which have proven to be adaptable to all testedelite soybean varieties. This method is equally adapted for directgenetic transformation into elite soybean cultivars, thus potentiallyavoiding the need for extensive cross-breeding between varieties.

The time period required for this method is greatly reduced compared toother Agrobacterium-mediated transformation protocols. The soybeanembryos are exposed to Agrobacterium transformation as soon as 6-14hours after seed imbibition, are co-cultured for one to four days, andare then subjected to post-transformation selection. Viablephenotypically positive soybean shoots can be collected 3 to 6 weeksfrom the initiation of the procedure. The entire R0 (primarytransformant) plant life cycle is not greatly longer than the minimumrequired for a soybean plant to grow to maturity in a greenhouse.

As with other Agrobacterium-mediated methods, the foreign geneticconstruction, or transgene, to be inserted into the soybean genome iscreated in vitro by normal techniques of recombinant DNA manipulations.The genetic construct is then transformed into the Agrobacterium strainfor delivery into the soybean cells. The Agrobacterium is non-oncogenic,and several such strains are now widely available. The foreign geneticconstruction includes a selectable marker gene. Several such selectablemarker genes are known, such as the gene for neomycin phosphotransferaseII (NPT II), which expresses an enzyme conferring resistance to theantibiotic kanamycin and the related antibiotics neomycin, paromomycin,gentamicin, and G418. However, a preferred type of selectable markergene is one of the genes conferring resistance to the herbicideglyphosate, such as the EPSP synthase gene described in U.S. Pat. No.5,633,435 or the glyphosate oxidoreductase gene described in U.S. Pat.No. 5,463,175.

The starting material for the transformation process is a soybean seed.The seed is first soaked for softening and then induced to initiategermination. The seeds are imbibed in water for approximately 3 minutesand then allowed to soften for up to 2 hours. The softening step is notrequired for every seed lot. High quality seeds need less softening thanlow quality seeds. The softening step is to help keep the meristemintact. The seeds are then put on germination media and permitted tobegin germination for a time period of about 6-24 hours, preferably forabout 6-14 hours, and most preferably for about 8-12 hours.

Then the embryo is excised from the seed, and any primary leaf tissuesare removed to expose the meristem of the soybean embryo.

For Agrobacterium-mediated gene transfer, wounding of the plant tissueis known to facilitate gene transfer. Therefore it is preferred, but notnecessary, that the embryonic meristem is wounded at this step of theprocess. Many methods of wounding can be used, including, for example,cutting, abrading, piercing, poking, penetration with fine particles orpressurized fluids, plasma wounding, application of hyperbaric pressure,or sonication. Wounding can be performed using objects such as, but notlimited to, scalpels, scissors, needles, abrasive objects, airbrush,particles, electric gene guns, or sound waves. Another alternative isvacuum infiltration. The procedure to this point will have typicallytaken 12-14 hours.

Preferably the wounding is done by sonication or plasma blast wounding.Sonication can be done in a bath sonicator or with a probe sonicator. Awetting agent, such as, but not limited to, Triton X-100 may or may notbe used. Sonication can be done for between about 5 sec and about 10min, preferably between about 5 seconds and about 40 seconds. Sonicationis preferably done in the presence of the Agrobacterium.

Plasma blast wounding is done using an electric gene gun. Embryos areplaced from 3-6 cm, preferably about 4 cm from the exposed electrodes,with 10 μL of water between them. A Plexiglass dome is then placed overthe chamber and a partial vacuum is applied concurrent with an influx ofhelium gas. A minimum of about 16,000 volts is discharged through thewater, vaporizing the water, and creating a plasma and shock waveengulfing the embryos. The embryos are then placed into theAgrobacterium culture.

The explants are then inoculated with the Agrobacterium culture for afew minutes to a few hours, typically about 0.5-3 hours. The excessmedia is drained and the Agrobacterium are permitted to co-cultivatewith the meristem tissue for several days, typically three days in thedark. During this step, the Agrobacterium transfers the foreign geneticconstruct into some cells in the soybean meristem.

Next the explants are transferred to a medium containing the selectionagent and appropriate antibiotics. This step is intended to terminate orat least retard the growth of the non-transformed cells and kill theremaining Agrobacterium cells. The length of culture depends, in part,on the toxicity of the selection agent to untransformed cells. Forglyphosate selection, a two-day culture is effective, but the length ofthis culture step is variable, extending from one to seven days. Forkanamycin selection, the explants are cultured from one to seven days.

Following this step, the meristems are placed in a medium conducive toshoot development for 3-7 days. The MSR medium used in the examplesbelow contains benzylaminopurine (BAP), a shoot-inducing hormone.Glyphosate itself has also been found to induce shoot formation insoybean. The term hormone also includes cell growth regulating compoundsthat induce shoot formation, including, but not limited to, IAA, NAA,IBA, cytokinins, auxins, kinetins, glyphosate, and thiadiazorun.Whichever hormonal treatment is used, the individual transformed cellsin a meristem give rise to transgenic sectors of tissue that areincorporated to a varying extent into the shoot arising directly fromthe explant. After culture on the MSR medium, the explants aretransferred to WPM-BAP (a medium suitable for shoot development) for 4-5weeks.

The elongated shoots are ready for harvest 3-6 weeks after the start ofthe entire transformation process. The shoots are evaluated forphenotypic regularity and health, and only shoots with elongated stems(approximately 1 inch) and full trifoliate leaf formation are harvested.The collected shoots are placed on a rooting medium to induce rootformation. Root formation takes approximately 1-4 weeks, following whichthe plants can be transferred to soil and grown to full maturity.Ideally, the rooting medium also contains the selection agent, to helpto terminate any non-transformants.

The R0 plants created by this technique are transgenic plants and areregularly recovered with quite reasonable yields. The number ofindependent germline plant lines recovered is usually in the singledigit percentage number range. Thus, a repeat of this procedure on 100planted soybean meristems would typically yield 1-10 independent linesof transgenic soybean.

EXAMPLES

The following examples further illustrate the present invention. Theyare in no way to be construed as a limitation in scope and meaning ofthe claims.

Methods and Materials

Media Preparation

Media used in the Agrobacterium-mediated transformation protocolemployed to develop transformed soybean plants were prepared usingstandard methods known to one skilled in the art. Media formulations maybe found in the cited references or in the Media Table (Table 3) thatfollows these examples.

Agrobacterium Preparation

Agrobacterium tumefaciens transformation vectors were constructed usingstandard molecular techniques known to those skilled in the art. Theseexamples used the plasmid constructs pMON21112, containing both the FMVCP4syn gene and the e35s GUS gene; pMON15737, containing FMV GUS, NOSNPTII, and FMV CP4syn; pMON36133, containing e35S NPTII, GFP; andpMON36152, containing e35S cre, FMV CP4. The FMV CP4 gene used inconstructing the plasmids is the promoter from Figwort Mosaic Virus(FMV) followed by the CP4syn gene, a synthetic gene encoding CP4 EPSPsynthase. See, U.S. Pat. No. 5,633,435, which is incorporated byreference herein. EPSP synthase, when expressed, confers a substantialdegree of glyphosate resistance upon the plant cell and plants generatedtherefrom. The e35s GUS gene is a β-glucuronidase gene, which istypically used as a histochemical marker, behind the e35S promoter. TheFMV GUS gene is the FMV promoter with GUS. The NOS NPTII gene is aneomycin phosphotransferase gene, which confers resistance to kanamycin,behind the promoter for the nopaline synthase gene (NOS). GFP is thegene for green fluorescence protein, which is a selectable marker.Overnight cultures of Agrobacterium strain containing the plasmid usedwere grown to log phase and then diluted to a final optical density of0.3 to 0.6.

Example 1

Transformation and Regeneration of Explants

Soybean seeds of cultivar A5403 were surface sterilized for threeminutes in 50% clorox. Seeds were germinated in liquid bean germinatingmedia (BGM) at a depth of two times the depth of the beans and incubatedovernight at 20° C. dark. The composition of BGM is provided in theMedia Table (Table 3).

Seed axis were prepared by removing the seed coat, breaking off thecotyledons and carefully removing primary leaf tissue to expose themeristematic region. Explants were then plated on OR media perpendicularto the surface with meristems away from the media and stored at 15° C.dark overnight. OR is a MS medium as modified by Barwale et al. (Plants167:473-481, 1986) plus 3 mg/L BAP, 200 mg/L Carbenicillin, 62.5 mg/LCefotaxime, and 60 mg/L Benomyl.

The next day explants were prepared for inoculation. Non-woundedexplants were placed directly into the Agrobacterium tumefaciensinoculum. Wounded explants, those with damage to the meristematictissue, were wounded by blasting with gold particles, scoring with ascalpel blade, poking, sonication, or piercing with fine needles. Vacuuminfiltration was used in addition to and as an alternative to otherwounding techniques. After one hour in inoculum, explants were placedwith meristems facing down on plates containing filter paper and 3-10 mLof standard co-culture media ( 1/10 B5 medium [Gamborg et al., Exp. CellRes., 50:151-158, 1968]). Plates were incubated in the dark at roomtemperature for three days.

After the transformation culture, explants were transferred to liquid ORmedia containing 0.2 mM glyphosate and incubated for ee days in the darkat 23-28° C. Following this stage, explants are removed from OR+0.2 mMglyphosate media and transferred to MSR+0.2 mM glyphosate and incubatedin the dark at 28° C. for seven days. MSR media is MS media as listedabove modified to include 0.4 mg/L BAP and 0.04 mg/L IBA (indole3-butyric acid). Then explants were transferred to plantcons (smallplastic containers used to culture small plants) containing woody plantmedium (WPM) (McCown & Lloyd, Proc. International Plant PropagationSoc., 30:421, 1981) minus BAP+0.075 mM glyphosate and incubated in thelight in growth rooms at 28° C. with a 16-hour light/8-hour darkphotoperiod. This step induced shoot formation, and shoots were observedfrom some cultured explants at this stage. Typically, explants weretransferred to fresh WPM medium every two weeks until harvest wascomplete.

After five to six weeks, the explants had grown such that phenotypepositive shoots could be pulled and rooted. These plants were then sentto the greenhouse to grow out and for further analysis.

Transformation Efficiency

Glyphosate selection indicated a transformation efficiency rate of 1-3%.Transformation efficiency was determined by comparing the number ofphenotypically normal plants that survived the selection protocol withthe number of explants initially prepared and inoculated. Table 1summarizes the data on transformation efficiency. All plants indicatedas germline positive correctly passed the transgenes on to their progenyby Mendelian inheritance. TABLE 1 ^(AGROBACTERIUM) TRANSFORMATION OFMERISTEMS Construct pMON21112(FMV CP4, e35s GUS), Cultivar A5403 TotalTotal Shoots Total Germline No. of Explant Sent to CP4+ to Date GermlineExperiment Explants Preparation Inoculation GH Shoots (Apr. 20, 1998)T.E. 58.1 100 Bombarded Vacuum 3 3 2/3 2% Infiltrate 58.2 100 ScoredStandard 1 0 0/1 0% (no vacuum) 58.3 100 Scored Standard 4 4 2/4 2% 58.4100 Pierced Standard 1 1 1 1% 58.5 100 Pierced Vacuum 2 2 2/2 2% 138.880 Pierced Standard 1 n/a 1/1 1.2%  

Example 2

Soybean seeds of cultivar A4922 were soaked in sterile distilled waterfor three minutes at room temperature then drained and left moist fortwo hours with periodic rolling. After two hours, the seeds were placedin BGM medium to twice the depth of the seeds, and the seeds wereincubated at room temperature in the dark.

At twelve hours from initiation of germination, the seed axes wereremoved from the seeds and placed in sterile distilled water forholding. The meristems were then wounded by puncturing with a needle,the wounding being repeated at all three meristems in each seed axis,the primary and the two secondary meristems associated with eachaxillary leaf primordia. The meristems were then inoculated with inducedAgrobacterium culture carrying the transgene, approximately 12-14 hoursafter initiation of the germination. After two hours, the meristems weredrained and placed in co-culture medium for three days of co-cultivationin the dark.

At day 4, the explants were placed in a petri dish with BGM, swirled andshaken for 1 to 2 hours with changes of BGM medium. Then the explantswere plated on OR medium with 0.075 mM glyphosate and cultured in thedark at room temperature for two days.

At day 6 the explants were transferred to MSR medium with 0.075 mMglyphosate and cultured in the dark for three days at room temperature.

At day 9, the explants were transferred to plantcons containing WPMminus BAP but with 0.075 mM glyphosate for shooting and were incubatedin the light at 28° C. The shoots that appeared were cut from themeristems from which they arose after 4 to 5 weeks. The shoots wererooted and cultivated to maturity in a greenhouse.

Out of the original 170 explants subjected to this procedure, eightphenotypically positive plants that were glyphosate resistant wererecovered. Analysis of R1 data confirms the presence of the insertedtransgenes.

EXAMPLE 3

Sonication Wounding and Kanamycin Selection

Soybean seeds are soaked in sterile distilled water for three minutes atroom temperature, drained, and left moist for two hours. BGM medium isadded after two hours to 2-3 times the depth of the seed volume andincubated at room temperature in the dark for six to eleven hours.

At eight to thirteen hours from initiation of germination, the seed axesare removed from seeds and held in sterile distilled water. Explants arerinsed with sterile distilled water, drained and divided into sets of50-300. Sets are placed into a vessel along with Agrobacteria (inducedor not induced) and can also include a wetting agent. Examples ofvessels include a 25 mL glass test tube along with 2 mL of Agrobacteriumor a 125 mL glass flask with 5-10 mL Agrobacterium.

Each vessel is then held in a sonicator with 500-1000 mL of distilledwater in the bath ±0.1% Triton X-100 and sonicated for 5-30 seconds inthe test tubes or for 20-40 seconds in the flask. Total inoculation timeranges from five minutes to three hours ±fresh Agrobacterium, ±vacuuminfiltration pulled multiple times or held at 25 inches Hg or shaking at0-120 RPM on an orbital shaker.

Explants are then co-cultured on one filter paper with one to sevenmilliliters of 1/10 B5 medium for two to four days at 23° C. in thedark. Following co-culture, explants can be rinsed with BGM and shakenon an orbital shaker for two hours to reduce bacterial load beforetransferring to the next stage.

Selection of transformed shoots is obtained through the use of kanamycinor glyphosate.

Kanamycin Selection

Either kanamycin sulfate or kanamycin nitrate may be used. Afterco-culture, explants are then transferred to solid OR media plus 0 to300 ppm kanamycin for one to seven days at 23° C., dark. Explants arethen transferred to WPM media minus BAP containing 50 to 300 ppmkanamycin and placed at 28° C., 16 hours light/8 hours dark photoperiod.Subcultures to WPM media with the same concentration of kanamycin orhigher are made one to three weeks later.

Shoots are pulled between three and six weeks post-inoculation. Shootsare rooted on BRM with 0 to 175 ppm kanamycin.

Glyphosate Selection

After co-culture, explants are transferred either to OR, OR/MSR, ordirectly to WPM media plus 0 to 2000 μM glyphosate. Explants can spendtwo to seven days on OR media or two to five days on OR plus two toseven days on MSR media. Explants are then transferred to WPM minus BAP.In some cases a fresh transfer to the same media is made after twoweeks.

Shoots are pulled between three and six weeks post-inoculation. Shootsare rooted on BRM (see Table 3) with 0 to 40 μM glyphosate.

Germlines are confirmed by testing leaf tissue of R1 plants by GUSassay, NPTII ELISA, CP4 ELISA, or PCR.

Table 2 shows transformation results for three different constructs andthree soybean varieties utilizing either kanamycin or glyphosateselection after sonication or poke wounding. Germline efficiencies(Total number of explants/Total number of germline transformants) rangefrom 0.5% to 3.3%. TABLE 2 Agrobacterium Transformation Using Sonicationand Poke Wounding Utilizing Glyphosate or Kanamycin SelectionConstructs: pMON15737 (FMV:GUS; NOS:NPTII; FMV:CP4syn) pMON36133(e35s:lox:modified NPTII:lox:GFP) pMON36152 (e35s:cre; FMV:arab EPSPTP:CP4 EPSPS) No. of Sonication Inoculation No. of Germline ExperimentConstruct Variety Explants Duration(sec) (hours) Selection Germlines TE4.2 pMON36133 A4922 106 30 1 kanamycin (SO4) 1 0.9% 60.6 pMON36133 A492233 5 1 + vacuum kanamycin (SO4) 1 3.0% 60.7 pMON36133 A4922 30 30 1 +vacuum kanamycin (SO4) 1 3.3% 92.5 pMON15737 A3244 92 15 1 + vacuumkanamycin (NO3) 1 1.1% 123.1 pMON15737 A3469 200  poked* 1.25 kanamycin(NO3) 1 0.5% 123.2 pMON15737 A3469 183 poked 1.25 + vacuum kanamycin(NO3) 1 0.5% 11.1 pMON36152 A4922 62 15 1 glyphosate 1 1.6% 39.1pMON36152 A4922 38 15 3 glyphosate 1 2.6% 68.1 pMON15737 A4922 105 151.5 glyphosate 2 1.9% 70.2 pMON15737 A4922 107 15 1.25 + vacuumglyphosate 1 0.9% 81.2 pMON15737 A4922 113 15 1.75 + vacuum glyphosate 10.9%*poked = single stab to center of all three meristems using a 4 flatshader tattoo needle

Example 4

Plasma Blast Wounding

Soybeans were germinated for 14 hours as described in Example 2.Wounding was done by suspending the embryos 4 cm from the exposedelectrodes of the electric gene gun. Discharge was set to 16,000 volts.A partial vacuum under helium gas was established in the blast chamber.The embryos were engulfed in the plasma and shock wave upon dischargingthe voltage through a 10 μL water droplet bridging the electrodes.

After wounding, embryos were incubated with Agrobacterium inoculumcontaining pMON15737 for 1.5 hours and selected with glyphosate asdescribed in Example 2. Of the seven targets of 12 embryos for thecontrol group, one phenotypic shoot was observed and did not root onglyphosate rooting medium. Of the seven targets of 12 embryos for thetreatment group, three phenotypic shoots were observed, one of theserooted and was sent to the greenhouse. It tested positive for GUSexpression in the vascular tissue, which indicates germlinetransformation, and had GUS positive seed, confirming transformation atthe RI generation. TABLE 3 MEDIA BEAN GERMINATION MEDIA (BGM 2.5%)COMPOUND: QUANTITY PER LITER BT STOCK #1 10 mL BT STOCK #2 10 mL BTSTOCK #3 3 mL BT STOCK #4 3 mL BT STOCK #5 1 mL SUCROSE 25 g Adjust topH 5.8.

Dispensed in 1 Liter Media Bottles, Autoclaved ADDITIONS PRIOR TO USE:PER 1 L CEFOTAXIME (50 mg/mL) 2.5 mL FUNGICIDE STOCK   3 mLBt Stock for Bean Germination Medium

Make and store each stock individually. Dissolve each chemicalthoroughly in the order listed before adding the next. Adjust volume ofeach stock accordingly. Store at 4° C. Bt Stock 1 (1 liter) KNO₃ 50.5 gNH₄NO₃ 24.0 g MgSO₄*7H₂O 49.3 g KH₂PO₄ 2.7 g Bt Stock 2 (1 liter)CaCl₂*2H₂O 17.6 g Bt Stock 3 (1 liter) H₃BO₃ 0.62 g MnSO₄₋H₂O 1.69 gZnSO₄—7H₂O 0.86 g KI 0.083 g NaMoO₄—2H₂O 0.072 g CuSO₄—5H₂O 0.25 mL of1.0 mg/mL stock CoCl₄—6H₂O 0.25 mL of 1.0 mg/mL stock Bt Stock 4 (1liter) Na₂EDTA 1.116 g FeSO₄7H₂O 0.834 g Bt Stock 5 (500 mL) Store in afoil wrapped container Thiamine-HCl 0.67 g Nicotinic Acid 0.25 gPyridoxine-HCl 0.41 g BRM MEDIA STOCK (for 4 L) MS Salts 8.6 gMyo-Inositol (Cell Culture Grade) .40 g Soybean Rooting Media VitaminStock 8 mL L-Cysteine (10 mg/mL) 40 mL Sucrose (Ultra Pure) 120 g pH 5.8Washed Agar 32 g ADDITIONS AFTER AUTOCLAVING: BRM/TSG Hormone Stock 20.0mL Ticarcillin/clavulanic acid (100 mg/mL Ticarcillin) 4.0 mL

SOY TISSUE CULTURE HORMONE PRE-MIXES MSR Pre-mixed Hormones Use 10.0 mLper liter Store dark at 4° C. Amount for 1 liter Amount for 20 liters 0.80 mL BAP (0.5 mg/mL)  16.0 mL BAP (0.5 mg/mL) 0.040 mL IBA (1.0mg/mL)  0.80 mL IBA (1.0 mg/mL)  9.16 mL SDW (sterile distilled water)183.2 mL SDW OR Pre-mixed Hormones Use 10.0 mL per liter. Store dark at4° C. Amount for 1 liter Amount for 30 liters  6.0 mL BAP (0.5 mg/mL)180.0 mL BAP (0.5 mg/mL) 0.037 mL NAA (1.0 mg/mL)  1.11 mL NAA (1.0mg/mL)  3.96 mL SDW 118.8 mL SDW WPM Pre-mixed Hormones Use 10.0 mL perliter Amount for 1 liter Amount for 50 liters 0.080 mL BAP (0.5 mg/mL) 4.0 mL BAP (0.5 mg/mL)  9.92 mL SDW 496.0 mL SDW Store dark at 4° C.BRM/TSG Hormone Stock Amount for 1 liter Amount for 40 liters 6.0 mL IAA(0.033 mg/mL) 240.0 mL IAA (0.033 mg/mL) 4.0 mL SDW 160.0 mL SDW Storedark at 4° C.

VITAMIN STOCK FOR SOYBEAN ROOTING MEDIA (1 liter) Glycine  1.0 gNicotinic Acid 0.25 g Pyridoxine HCl 0.25 g Thiamine HCl 0.05 g

Dissolve one ingredient at a time, bring to volume, store infoil-covered bottle in refrigerator for no more than one month. 3× MINORMS SALTS STOCK (1 liter) H₃BO₃ 1.86 g MnSO₄—H₂O 5.07 g ZnSO₄—7H₂O 2.58 gKI 0.249 g NaMoO—2H₂O 0.075 g CuSO₄—5H₂O Stock (1.0 mg/mL) 7.5 μLCoCl₂—6H₂O Stock (1.0 mg/mL) 7.5 μL

Dissolve one chemical at a time, adjust volume, store in refrigerator.FUNGICIDE STOCK (100 mL) chlorothalonile (75% WP) 1.0 g benomyl (50% WP)1.0 g captan (50% WP) 1.0 gAdd to 100 mL of sterile distilled water.Shake well before using.Store 4° C. dark for no more than one week.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1-10. (canceled)
 11. A method of producing a transformed soybean cellcomprising: (a) initiating the germination of a soybean seed; (b)substantially removing at least one cotyledon from the germinating seed;(c) exposing at least a portion of the meristematic region in thegerminating seed; (d) exposing one or more cells in the meristematicregion to a disarmed Agrobacterium vector comprising a heterologousgenetic construct, wherein said genetic construct comprises a selectablemarker gene, and said genetic construct is transferred into at least onecell in the meristematic region; (e) culturing the cells of themeristematic region in the presence of a selection agent to identify atransformed soybean cell that contains the genetic construct.
 12. Themethod of claim 11, wherein the selectable marker gene confersresistance to an antibiotic or a herbicide.
 13. The method of claim 11,wherein the selectable marker gene confers resistance to glyphosate. 14.The method of claim 11, wherein the selectable marker gene encodes anEPSP synthase.
 15. The method of claim 14, wherein the EPSP synthaseprotein is the CP4 protein.
 16. The method of claim 11, wherein theselection agent is an herbicide.
 17. The method of claim 16, wherein theherbicide is glyphosate.
 18. The method of claim 11, wherein theselectable marker gene confers resistance to kanamycin.
 19. The methodof claim 11, wherein the selectable marker gene encodes a neomycinphosphotransferase II (nptII) protein.
 20. The method of claim 11,wherein the selection agent is kanamycin.
 21. The method of claim 11,further comprising: (f) inducing formation of one or more shoots fromthe meristematic region, wherein said shoot comprises at least onetransformed cell, to produce a transgenic soybean shoot.
 22. The methodof claim 21, wherein step (f) comprises exposing said meristematicregion to at least one shoot inducing hormone.
 23. The method of claim22, wherein the at least one shoot inducing hormone is selected from thegroup consisting of BAP, IBA, IAA, NAA, cytokinins, auxins, kinetins,glyphosate, and thiadiazorun.
 24. The method of claim 21, wherein step(f) comprises exposing said meristematic region to a media containingBAP and IBA.
 25. A method of producing a fertile transgenic soybeanplant comprising: (a) initiating the germination of a soybean seed; (b)substantially removing at least one cotyledon from the germinating seed;(c) exposing at least a portion of the meristematic region in thegerminating seed; (d) exposing one or more cells in the meristematicregion to a disarmed Agrobacterium vector comprising a heterologousgenetic construct, wherein said genetic construct comprises a selectablemarker gene, and said genetic construct is transferred into at least onecell in the meristematic region; (e) culturing the cells of themeristematic region in the presence of a selection agent in a mannerthat identifies a transformed soybean cell that contains the geneticconstruct. (f) inducing formation of one or more shoots from themeristematic region, wherein said shoot comprises at least onetransformed cell, to produce a transgenic soybean shoot. (g) cultivatingthe transgenic soybean shoot into a fertile transgenic soybean plant.26. The method of claim 25, wherein step (g) comprises inducing rootformation from the transgenic soybean shoot.
 27. The method of claim 25,wherein step (f) comprises exposing said meristematic region to at leastone shoot inducing hormone.
 28. The method of claim 22, wherein the atleast one shoot inducing hormone is selected from the group consistingof BAP, IBA, IAA, NAA, cytokinins, auxins, kinetins, glyphosate, andthiadiazorun.
 29. The method of claim 25, wherein step (f) comprisesexposing said meristematic region to a media containing BAP and IBA. 30.The method of claim 25, wherein the selectable marker gene confersresistance to an antibiotic or a herbicide.
 31. The method of claim 25,wherein the selectable marker gene confers resistance to glyphosate. 32.The method of claim 25, wherein the selectable marker gene encodes anEPSP synthase.
 33. The method of claim 32, wherein the EPSP synthaseprotein is the CP4 protein.
 34. The method of claim 25, wherein theselection agent is an herbicide.
 35. The method of claim 34, wherein theherbicide is glyphosate.
 36. The method of claim 25, wherein theselectable marker gene confers resistance to kanamycin.
 37. The methodof claim 25, wherein the selectable marker gene encodes a neomycinphosphotransferase II (nptII) protein.
 38. The method of claim 25wherein the selection agent is kanamycin.
 39. The method of claim 25,wherein said fertile transgenic soybean plant is cultivated to produceseed.
 40. The method of claim 39, wherein said seed are cultivated toproduce progeny soybean plants.
 41. The method of claim 40, wherein saidprogeny soybean plants are cultivated to produce progeny seed.
 42. Themethod of claim 41, wherein said progeny seed is cultivated to produce asubsequent generation of soybean plants.
 43. The method of claim 11,wherein the exposed meristematic region is wounded after step (c). 44.The method of claim 43, wherein the wounding is caused by ultrasonicwaves, a plasma blast discharge, or by puncturing the soybean explantwith a needle, other sharp object, or an abrasive object.
 45. The methodof claim 25, wherein the exposed meristematic region is wounded afterstep (c).
 46. The method of claim 45, wherein the wounding is caused byultrasonic waves, a plasma blast discharge, or by puncturing the soybeanexplant with a needle, other sharp object, or an abrasive object.